Biological characteristics of Cordyceps militaris single mating-type strains.

Cordyceps militaris has been extensively cultivated as a model cordyceps species for commercial purposes. Nevertheless, the problems related to strain degeneration and breeding technologies remain unresolved. This study assessed the physiology and fertility traits of six C. militaris strains with distinct origins and characteristics, focusing on single mating-type strains. The results demonstrated that the three identified strains (CMDB01, CMSY01, and CMJB02) were single mating-type possessing only one mating-type gene (MAT1-1). In contrast, the other three strains (CMXF07, CMXF09, and CMMS05) were the dual mating type. The MAT1-1 strains sourced from CMDB01, CMSY01, and CMJB02 consistently produced sporocarps but failed to generate ascospores. However, when paired with MAT1-2 strains, the MAT1-1 strains with slender fruiting bodies and normal morphology were fertile. The hyphal growth rate of single mating-type strains (CMDB01, CMSY01, and CMJB02) typically surpassed that of dual mating-type strains (CMXF07, CMXF09, and CMMS05). The growth rates of MAT1-2 and MAT1-1 strains were proportional to their ratios, such that a single mating-type strain with a higher ratio exhibited an increased growth rate. As C. militaris matured, the adenosine content decreased. In summary, the C. militaris strains that consistently produce sporocarps and have a single mating type are highly promising for production and breeding.

A rho-type GTPase activating protein affects the growth and development of Cordyceps cicadae.

Cordyceps cicadae is recognized for its medicinal properties, attributed to bioactive constituents like polysaccharides and adenosine, which have been shown to improve kidney and liver functions and possess anti-tumor properties. Rho GTPase activating proteins (Rho GAPs) serve as inhibitory regulators of Rho GTPases in eukaryotic cells by accelerating the GTP hydrolysis of Rho GTPases, leading to their inactivation. In this study, we explored the function of the CcRga8 gene in C. cicadae, which encodes a Rho-type GTPase activating protein. Our study found that the knockout of CcRga8 resulted in a decrease in polysaccharide levels and an increase in adenosine concentration. Furthermore, the mutants exhibited altered spore yield and morphology, fruiting body development, decreased infectivity, reduced resistance to hyperosmotic stress, oxidative conditions, and cell wall inhibitors. These findings suggest that CcRga8 plays a crucial role in the development, stress response, and bioactive compound production of C. cicadae.

Ethanolic extract from fruiting bodies of Cordyceps militaris HL8 exhibits cytotoxic activities against cancer cells, skin pathogenic yeasts, and postharvest pathogen Penicillium digitatum.

Cordyceps militaris is a well-known medicinal mushroom in Asian countries. This edible fungus has been widely exploited for traditional medicine and functional food production. C. militaris is a heterothallic fungus that requires both the mating-type loci, MAT1-1 and MAT1-2, for fruiting body formation. However, recent studies also indicated two groups of C. militaris, including monokaryotic strains carrying only MAT1-1 in their genomes and heterokaryotic strains harboring both MAT1-1 and MAT1-2. These strain groups are able to produce fruiting bodies under suitable cultivating conditions. In previous work, we showed that monokaryotic strains are more stable than heterokaryotic strains in fruiting body formation through successive culturing generations. In this study, we report a high cordycepin-producing monokaryotic C. militaris strain (HL8) collected in Vietnam. This strain could form normal fruiting bodies with high biological efficiency and contain a cordycepin content of 14.43 mg/g lyophilized fruiting body biomass. The ethanol extraction of the HL8 fruiting bodies resulted in a crude extract with a cordycepin content of 69.15 mg/g. Assays of cytotoxic activity on six human cancer cell lines showed that the extract inhibited the growth of all these cell lines with the IC50 values of 6.41-11.51 µg/mL. Notably, the extract significantly reduced cell proliferation and promoted apoptosis of breast cancer cells. Furthermore, the extract also exhibited strong antifungal activity against Malassezia skin yeasts and the citrus postharvest pathogen Penicillium digitatum. Our work provides a promising monokaryotic C. militaris strain as a bioresource for medicine, cosmetics, and fruit preservation.

Improvement of nucleotide content of Cordyceps tenuipes by Schisandra chinensis: fermentation process optimization and application prospects.

Nucleotides are important components and the main indicators for judging Cordyceps quality. In this paper, the mixed fermentation process of Schisandra chinensis and Cordyceps tenuipes was systematically studied, and it was proposed that the fermentation products aqueous extract (S-ZAE) had antioxidant activity and anti-AChE ability. Herein, the results of a single factor showed that S. chinensis, yeast extract, inoculum amount, and pH had significant effects on nucleotide synthesis. The fermentation process optimization results were 3% glucose, 0.25% KH2PO4, 2.1% yeast extract, and S. chinensis 0.49% (m/v), the optimal fermentation conditions were 25℃, inoculum 5.8% (v/v), pH 3.8, 6 d. The yield of total nucleotides in the scale-up culture was 0.64 ± 0.027 mg/mL, which was 10.6 times higher than before optimization. S-ZAE has good antioxidant and anti-AChE activities (IC50 0.50 ± 0.050 mg/mL). This fermentation method has the advantage of industrialization, and its fermentation products have the potential to become good functional foods or natural therapeutic agents.

A novel betapartitivirus isolated from Cordyceps militaris, an edible-medicinal mushroom.

In this study, we identified a novel partitivirus, named "Cordyceps militaris partitivirus 1" (CmPV1), in Cordyceps militaris strain RCEF7506. The complete genome of CmPV1 comprises two segments, dsRNA1 and dsRNA2, each encoding a single protein. dsRNA1 (2,206 bp) encodes an RNA-dependent RNA polymerase (RdRp), and dsRNA2 (2,256 bp) encodes a coat protein (CP). Sequence analysis revealed that dsRNA1 has the highest similarity to that of Bipolaris maydis partitivirus 2 (BmPV2), whereas dsRNA2 shows the highest similarity to human blood-associated partitivirus (HuBPV). Phylogenetic analysis based on RdRp sequences suggests that CmPV1 is a new member of the genus Betapartitivirus of the family Partitiviridae. This is the first documentation of a betapartitivirus infecting the entomopathogenic fungus C. militaris.

Identification of a novel member of the genus Laulavirus (family Phenuiviridae) from the entomopathogenic ascomycete fungus Cordyceps javanica.

The virus family Phenuiviridae (order Hareavirales, comprising segmented negative-sense single stranded RNA viruses) has highly diverse members that are known to infect animals, plants, protozoans, and fungi. In this study, we identified a novel phenuivirus infecting a strain of the entomopathogenic fungus Cordyceps javanica isolated from a small brown plant hopper (Laodelphax striatellus), and this virus was tentatively named "Cordyceps javanica negative-strand RNA virus 1" (CjNRSV1). The CjNRSV1 genome consists of three negative-sense single stranded RNA segments (RNA1-3) with lengths of 7252, 2401, and 1117 nt, respectively. The 3'- and 5'-terminal regions of the RNA1, 2, and 3 segments have identical sequences, and the termini of the RNA segments are complementary to each other, reflecting a common characteristic of viruses in the order Hareavirales. RNA1 encodes a large protein (∼274 kDa) containing a conserved domain for the bunyavirus RNA-dependent RNA polymerase (RdRP) superfamily, with 57-80% identity to the RdRP encoded by phenuiviruses in the genus Laulavirus. RNA2 encodes a protein (∼79 kDa) showing sequence similarity (47-63% identity) to the movement protein (MP, a plant viral cell-to-cell movement protein)-like protein (MP-L) encoded by RNA2 of laulaviruses. RNA3 encodes a protein (∼28 kDa) with a conserved domain of the phenuivirid nucleocapsid protein superfamily. Phylogenetic analysis using the RdRPs of various phenuiviruses and other unclassified phenuiviruses showed CjNRSV1 to be grouped with established members of the genus Laulavirus. Our results suggest that CjNRSV1 is a novel fungus-infecting member of the genus Laulavirus in the family Phenuiviridae.

Tissue distribution of metabolites in Cordyceps cicadae determined by DESI-MSI analysis.

In this paper, we establish an in situ visualization analysis method to image the spatial distribution of metabolites in different parts (sclerotium, coremium) and different microregions of Cordyceps cicadae (C. cicadae) to achieve the in situ visual characterization of tissues for a variety of metabolites such as nucleosides, amino acids, polysaccharides, organic acids, fatty acids, and so on. The study included LC-MS chemical composition identification, preparation of C. cicadae tissue sections, DEDI-MSI analysis, DESI combined with Q-TOF/MS to obtain high-resolution imaging of mass-to-charge ratio and space, imaging of C. cicadae in positive-negative ion mode with a spatial resolution of 100 µm, and localizing and identifying its chemical compositions based on its precise mass. A total of 62 compounds were identified; nucleosides were mainly distributed in the coremium, L-threonine and DL-isoleucine, and other essential amino acids; peptides were mainly distributed in the sclerotium of C. cicadae; and the rest of the amino acids did not have a clear pattern; sugars and sugar alcohols were mainly distributed in the coremium of C. cicadae; organic acids and fatty acids were distributed in the nucleus of C. cicadae more than in the sclerotium, and the mass spectrometry imaging method is established in the research. The mass spectrometry imaging method established in this study is simple and fast and can visualize and analyse the spatial distribution of metabolites of C. cicadae, which is of great significance in characterizing the metabolic network of C. cicadae, and provides support for the quality evaluation of C. cicadae and the study of the temporal and spatial metabolic network of chemical compounds.

Bioinformatic Approaches Identify Hybrid Antibiotics against Tuberculosis via d-Amino Acid-Activating Adenylation Domains from Cordyceps militaris.

The development of tuberculosis (TB) therapy has been marked by the discovery of natural-product-derived streptomycin, followed by the introduction of NP-derived rifampicin, representing a significant milestone in the history of TB management. However, TB remains a global challenge, with the emergence of multidrug-resistant Mycobacterium tuberculosis highlighting the need for novel therapeutic agents. In this study, a bioinformatic approach was employed to investigate d-amino acid-activating adenylation domains, leading to the identification of cordysetin A (1), a novel trans-decalin tetramic acid antibiotic from the ascomycete fungi Cordyceps militaris. Cordysetin A (1) exhibits considerable activity against M. tuberculosis in vitro and in vivo while maintaining low cytotoxicity. These results reveal that the d-configuration of the amino acid within this hybrid polyketide-nonribosomal antibiotic is crucial for preserving its anti-tuberculosis efficacy. These findings emphasize the significant translational potential of cordysetin A as a promising candidate for TB treatment, furthering our understanding of bioinformatic approaches in the development of effective anti-tuberculosis agents.

Isolation and target identification of anti-renal fibrosis compounds from Cordyceps militaris.

Four undescribed compounds including one aromatic glucoside derivative, cordyceglycoside A (1), one new isoleucine derivative inner salt, cordycepisosalt A (2), a rare four-membered lactam, cinerealactam B (3), and one sesquiterpene derivative, cordycepsetp A (4), together with six known compounds were isolated from Cordyceps militaris. The structures including absolute configurations of these new compounds, were unambiguously elucidated by spectroscopic data analysis and single crystal X-ray diffraction. Biological evaluation of compounds 1-4 showed that 3 displays anti-renal fibrotic activities in TGF-ß1 induced NRK-52e cells. Furthermore, DARTS coupled with LC-MS/MS analysis was used to identify candidate target proteins for 3. Subsequently, C1qbp knockdown using siRNA allowed us to validate the target protein of 3.

Fermented Ophiocordyceps sinensis mycelium products for preventing contrast-associated acute kidney injury: a systematic review of randomized controlled trials.

BACKGROUND: To evaluate the efficacy, effectiveness and safety of fermented Ophiocordyceps sinensis mycelium (FOSM) products for preventing contrast-associated acute kidney injury (CA-AKI). METHODS: Randomized controlled trials were searched from four Chinese and four English electronic databases and three clinical trial registries up to July 2023. Methodological quality was assessed by using the Cochrane risk-of-bias tool 2.0. Risk difference (RD) or risk ratio (RR) and mean difference (MD) were calculated along with the 95% confidence intervals (CIs). RESULTS: Fourteen trials testing three types of FOSM products (Bailing, Zhiling, and Jinshuibao capsules) involving 1271 participants injected contrast agents were included. For the risk of bias, all trials were rated as some concerns. Compared with routine preventive procedure (RPP) (saline hydration and alprostadil), FOSM products plus RPP showed beneficial effects in reducing the incidence of CA-AKI (14.62% and 5.35%, respectively; RD -0.06, 95% CI -0.09 to -0.03). Subgroup analysis showed that Bailing/Jinshuibao plus RPP demonstrated lower incidence of CA-AKI compared to RPP. However, there was no statistically significant difference between Zhiling with RPP and RPP in the incidence of CA-AKI. Additionally, only when FOSM products were taken before injection of the contrast, it was superior to RPP in reducing the incidence of CA-AKI. There was no statistical difference in adverse events between these two groups. CONCLUSIONS: Low certainty evidence suggests that preventive oral use of FOSM products as an adjuvant agent was safe and might decrease the incidence of CA-AKI. However, high-quality placebo-controlled trials are needed to confirm its benefit.

Metabolite profiling and antioxidant capacity of natural Ophiocordyceps gracilis and its cultures using LC-MS/MS-based metabolomics: Comparison with Ophiocordyceps sinensis.

INTRODUCTION: Ophiocordyceps gracilis is an entomopathogenic fungus and a precious traditional Chinese medicine with similar medicinal properties to Ophiocordyceps sinensis. However, information on the metabolite profiles of natural O. gracilis and its cultures is lacking, which limits their utilization. OBJECTIVE: The metabolic variations and antioxidant activities of O. gracilis cultures and natural O. gracilis were analyzed to evaluate the nutritional and medicinal value of O. gracilis and its cultures. METHOD: The metabolite profiles of O. gracilis cultures (fruiting bodies and aerial mycelia), natural O. gracilis, and natural O. sinensis were compared by LC-MS/MS coupled with multivariate data analysis. Furthermore, their antioxidant activities were evaluated based on their DPPH• , ABTS•+ , and • OH scavenging abilities. RESULTS: A total of 612 metabolites were identified, and the metabolic compositions of the four Cordyceps samples were similar, with differences observed in the levels of some metabolites. There were 126 differential metabolites between natural O. gracilis and natural O. sinensis, among which fatty acids, carbohydrates, and secondary metabolites are predominant in natural O. gracilis. Furthermore, 116 differential metabolites between O. gracilis cultures and natural Cordyceps were identified, with generally higher levels in O. gracilis cultures than in natural Cordyceps. O. gracilis cultivated fruiting bodies exhibited the strongest antioxidant capacity among Cordyceps samples. Additionally, 46 primary and 24 secondary differential metabolites contribute to antioxidant activities. CONCLUSION: This study provides a reference for the application of natural O. gracilis and its cultures in functional food and medicine from the perspective of metabolites and antioxidant capacity.

Enhanced polysaccharide production through quorum sensing system in Cordyceps militaris.

This study aimed to enhance extracellular polysaccharide (EPS) production in Cordyceps militaris by constructing a quorum sensing (QS) system to regulate the expression of biosynthetic enzyme genes, including phosphoglucomutase, hexokinase, phosphomannomutase, polysaccharide synthase, and UDP-glucose 4-epimerase genes. The study found higher EPS concentrations in seven recombinant strains compared to the wild-type C. militaris, indicating that the overexpression of key enzyme genes increased EPS production. Among them, the CM-pgm-2 strain exhibited the highest EPS production, reaching a concentration of 3.82 ± 0.26 g/L, which was 1.52 times higher than the amount produced by the wild C. militaris strain. Additionally, the regulatory effects of aromatic amino acids on the QS system of the CM-pgm-2 strain were investigated. Under the influence of 45 mg/L tryptophan, the EPS production in CM-pgm-2 reached 4.75 ± 0.20 g/L, representing a 1.90-fold increase compared to wild C. militaris strains. This study provided an effective method for the large-scale production of EPSs in C. militaris, and opened up new avenues for research into fungal QS mechanisms.

NAD-Driven Sirtuin Activation by Cordyceps sinensis Extract: Exploring the Adaptogenic Potential to Promote Skin Longevity.

In recent years, there has been increasing interest in utilizing Traditional Chinese Medicine principles and natural bioactive compounds to combat age-related ailments and enhance longevity. A Cordyceps sinensis mycelium hydroethanolic extract (CsEx), which was standardized in cordycepin and adenosine using UHPLC-DAD, was investigated for its adaptogenic properties using in vitro assays and a double-blind, placebo-controlled clinical trial involving 40 subjects. The CsEx demonstrated activity at a concentration of 0.0006%, significantly increasing sirtuin expression (SirT1: +33%, SirT3: +10%, SirT6: +72%, vs. CTR, p < 0.05) and NAD+ synthesis in HaCat cells (+20% vs. CTR, p < 0.001). Moreover, the CsEx boosted ATP production by 68% in skin cells, correlating with higher skin energy values (+52.0% at D28, p < 0.01) in the clinical trial. Additionally, CsEx notably reduced cytosolic reactive oxygen species (ROS) by 30% in HaCaT cells (p < 0.05) and enhanced collagen production both in vitro (+69% vs. CTR, p < 0.01) and in vivo (+10% vs. D0, p < 0.01), confirmed by ultrasound examination. Furthermore, CsEx's stimulation of fibroblasts, coupled with its antioxidant and energizing properties, led to a significant reduction in wrinkles by 28.0% (D28, p < 0.001). This study underscores Cordyceps sinensis hydroethanolic extract's potential in regulating skin cell energy metabolism and positively influencing the mechanisms associated with skin longevity control.

Polysaccharides as Quality Marker to Rapid Profile for Ophiocordyceps sinensis by PXRD.

BACKGROUND: Ophiocordyceps sinensis has long been recognized as a mysterious and valuable traditional Chinese medicine but there has been little research on quality markers for O. sinensis. PURPOSE: This study looked into the potential of using powder X-ray diffractometry (PXRD) to analyze polysaccharides as a quality marker for O. sinensis. STUDY DESIGN: There were 16 different habitats of O. sinensis collected in Qinghai, Gansu, Sichuan, Yunnan, and Tibet. In addition, five different types of Cordyceps species were collected. The characteristic diffraction peaks of O. sinensis were determined and then matched with the characteristic diffraction peaks of intracellular polysaccharides obtained from O. sinensis to determine the attribution relationship of the characteristic diffraction peaks. METHODS: O. sinensis powder's X-ray diffraction pattern is determined by its composition, microcrystalline crystal structure, intramolecular bonding mechanism, and molecular configuration. After fractionation and alcohol precipitation of crude intracellular polysaccharide, mycelium crude intracellular polysaccharide (MCP) and fruiting body crude intracellular polysaccharide (FCP) were obtained and the fingerprint of O. sinensis was identified by the specific characteristic peaks of the X-ray diffraction pattern from intracellular polysaccharide. RESULTS: The results indicated that the PXRD patterns of different populations of O. sinensis were overlaid well with 18 characteristic diffraction peaks obtained by microcrystalline diffraction. Moreover, the powder diffractograms as a fingerprint provided a practical identification of O. sinensis from other Cordyceps species. In addition, we detected that the powder diffractograms of intracellular polysaccharide MCP and MCP75 could be coupled with the PXRD of O. sinensis. Specifically, 18 characteristic diffraction peaks were identified as coming from MCP and MCP75 according to those interplanar crystal spacing, which matched well with those of PXRD of O. sinensis. CONCLUSIONS: PXRD spectra combined with an updated multivariable discriminant model were found to be an efficient and sensitive method for O. sinensis quality control. According to the findings of this study, PXRD should be further investigated for quality control assessments and plant extract selection trials.

Immunomodulatory Effect of Cordyceps militaris Polysaccharide on RAW 264.7 Macrophages by Regulating MAPK Signaling Pathways.

Polysaccharide is one of the principal bioactive components found in medicinal mushrooms and has been proven to enhance host immunity. However, the possible mechanism of immunomodulatory activity of Cordyceps militaris polysaccharide is not fully understood. Hot water extraction and alcohol precipitation, DEAE-Sephadex A-25 chromatography, and Sephadex G-100 chromatography were used to isolate polysaccharide from C. militaris. A high-molecular-weight polysaccharide isolated from C. militaris was designated as HCMP, which had an Mw of 6.18 × 105 Da and was composed of arabinose, galactose, glucose, mannose, and xylose in a mole ratio of 2.00:8.01:72.54:15.98:1.02. The polysaccharide content of HCMP was 91.2% ± 0.16. The test in vitro showed that HCMP activated mouse macrophage RAW 264.7 cells by enhancing phagocytosis and NO production, and by regulating mRNA expressions of inflammation-related molecules in RAW 264.7 cells. Western blotting revealed that HCMP induced the phosphorylation of mitogen-activated protein kinases (MAPKs). Moreover, using inhibitors of MAPKs decreased the mRNA levels of inflammation-related molecules induced by HCMP. These data evidenced that the immunomodulatory effect of HCMP on RAW 264.7 macrophages was mediated via the MAPK signaling pathway. These findings suggested that HCMP could be developed as a potent immunomodulatory agent for use in functional foods and dietary supplements.

Verification of In Vitro Anticancer Activity and Bioactive Compounds in Cordyceps Militaris-Infused Sweet Potato Shochu Spirits.

Many liqueurs, including spirits infused with botanicals, are crafted not only for their taste and flavor but also for potential medicinal benefits. However, the scientific evidence supporting their medicinal effects remains limited. This study aims to verify in vitro anticancer activity and bioactive compounds in shochu spirits infused with Cordyceps militaris, a Chinese medicine. The results revealed that a bioactive fraction was eluted from the spirit extract with 40% ethanol. The infusion time impacted the inhibitory effect of the spirit extract on the proliferation of colon cancer-derived cell line HCT-116 cells, and a 21-day infusion showed the strongest inhibitory effect. Furthermore, the spirit extract was separated into four fractions, A-D, by high-performance liquid chromatography (HPLC), and Fractions B, C, and D, but not A, exerted the effects of proliferation inhibition and apoptotic induction of HCT-116 cells and HL-60 cells. Furthermore, Fractions B, C, and D were, respectively, identified as adenosine, cordycepin, and N6-(2-hydroxyethyl)-adenosine (HEA) by comprehensive chemical analyses, including proton nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy (FT-IR), and electrospray ionization mass spectrometry (ESI-MS). To better understand the bioactivity mechanisms of cordycepin and HEA, the agonist and antagonist tests of the A3 adenosine receptor (A3AR) were performed. Cell viability was suppressed by cordycepin, and HEA was restored by the A3AR antagonist MR1523, suggesting that cordycepin and HEA possibly acted as agonists to activate A3ARs to inhibit cell proliferation. Molecular docking simulations revealed that both adenosine and cordycepin bound to the same pocket site of A3ARs, while HEA exhibited a different binding pattern, supporting a possible explanation for the difference in their bioactivity. Taken together, the present study demonstrated that cordycepin and HEA were major bioactive ingredients in Cordyceps militaries-infused sweet potato shochu spirits, which contributed to the in vitro anticancer activity.

Improvement of the production and quality of Cordyceps javanica conidia for the control of Diaphorina citri adults.

This study aimed to evaluate the use of palm kernel meal (PKM) in the traditional solid-state fermentation system to improve the production and quality of Cordyceps javanica conidia. The impact of PKM was determined by measuring conidia yield, viability, hydrophobicity, shelf life, and conidia pathogenicity against Diaphorina citri adults. By supplementing rice grains with 5% palm kernel meal increased the conidial yield by up to 40%, without compromising conidia viability and hydrophobicity. In addition, conidia caused higher levels of mortality by mycosis against D. citri adults (90%), relative to conidia harvested from rice (52%). The conidia recovered from rice/palm kernel meal mixtures also retained viability greater than 90% after storage for 10 months at 4 °C, while the conidia produced on rice reached 80%. Thus, conidia produced in the presence of palm kernel meal can be consumed immediately or in the medium term. Some process advantages of the palm kernel meal as co-substrate in the traditional production system of C. javanica are also mentioned. These results are attractive for improving the mycoinsecticide production process, with excellent cost-benefit and minimal changes in infrastructure and process.

Entomopathogenic fungi in crops protection with an emphasis on bioactive metabolites and biological activities.

Plant pathogens with their abundance are harmful and cause huge damage to different agricultural crops and economy of a country as well as lead towards the shortage of food for humans. For their management, the utilization of entomopathogenic fungi is an eco-friendly technique, sustainable to the environment, safe for humans and has promising effect over chemical-based pesticides. This process requires a biochemical mechanism, including the production of enzymes, toxins, and other metabolites that facilitate host infection and invasion. Essential enzymes such as chitinase, proteinase, and lipase play a direct role in breaking down the host cuticle, the primary barrier to EPF (Entomopathogenic Fungi) infection. Additionally, secondary metabolites such as destruxins in Metarhizium, beauvericin in Beauveria, hirsutellides in Hirsutella, isarolides in Isaria, cordyols in Cordyceps, and vertihemipterins in Verticillium, among others, act both directly and indirectly to disable the defense mechanisms of insect hosts, thereby accelerating the EPF infection process. The chemical composition of these secondary metabolites varies, ranging from simple non-peptide pigments such as oosporine to highly complex piperazine derivatives such as vertihemiptellides. The biocontrol efficacy of EPF is extensively studied, with numerous fungal strains commercially available on a large scale for managing arthropod pests. This review emphasizes the role of proteins and enzymes against crop pathogens, detailing their mode of action, and describing the metabolites from entomopathogenic fungi and their biological activities. In doing so, these findings contribute to establishing a symbiotic equilibrium between agricultural productivity and environmental conservation.

Recognition of antitussive components in Farfarae Flos based on grey relational analysis and partial least squares regression. / åŸºäºŽç°è‰²å ³è”åº¦åˆ†æžå’Œåæœ€å°äºŒä¹˜å›žå½’è¾¨è¯†æ¬¾å†¬èŠ±æ­¢å’³æ´»æ€§æˆåˆ†.

OBJECTIVES: Farfarae Flos has the effect of cough suppression and phlegm elimination, with cough suppression as the main function. Studies have revealed that certain components of Farfarae Flos may be related to its cough suppressant effect, and some components have been confirmed to have cough suppressant activity. However, the antitussive material basis of Farfarae Flos has not been systematically elucidated. This study aims to elucidate the group of active ingredients in Farfarae Flos with cough suppressant activity by correlating the high performance liquid chromatography (HPLC) fingerprint of Farfarae Flos extract with its cough suppressant activity. METHODS: HPLC was used to establish the fingerprint profiles of 10 batches of Farfarae Flos extract and obtain their chemical composition data. Guinea pigs were selected as experimental animals and the citric acid-induced cough model was used to evaluate the antitussive efficacy data of 10 batches of Farfarae Flos extract. SPF-grade healthy male Hartley guinea pigs were randomly divided into the S1 to S10 groups, a positive control group, and a blank control group (12 groups in total), with 10 guinea pigs in each group. The S1 to S10 groups were respectively administered Farfarae Flos extract S1 to S10 (4 g/kg), the positive control group was administered pentoverine citrate (10 mg/kg), and the blank control group was administered purified water. Each group received continuous oral administration for 5 days. The guinea pigs were placed in 5 L closed wide-mouth bottles, and 17.5% citric acid was sprayed into the bottle with an ultrasonic atomizer at the maximum spray intensity for 0.5 minutes. The cough latency period and cough frequency in 5 minutes were recorded for each guinea pig. Grey relational analysis (GRA) and partial least squares regression (PLSR) were used to conduct spectral-effect correlation analysis of the chemical composition data of Farfarae Flos extract and the antitussive efficacy data, and predict the group of active ingredients in Farfarae Flos with antitussive activity. The bioequivalence verification was conducted to verify the predicted group of active ingredients in Farfarae Flos with antitussive activity: SPF-grade healthy male Hartley guinea pigs were randomly divided into a S9 group, an active ingredient group, a positive control group, and a blank control group (4 groups in total), with 10 guinea pigs in each group. The S9 group was administered Farfarae Flos extract S9 (4 g/kg), the active ingredient group was administered the predicted combination of antitussive active ingredients (dose equivalent to 4 g/kg of Farfarae Flos extract S9), the positive control group was administered pentoverine citrate (10 mg/kg), and the blank control group was administered purified water. Each group received continuous oral administration for 5 days, and animal modeling and observation of efficacy indicators were the same as above. RESULTS: The HPLC fingerprint of 10 batches of Farfarae Flos extract was established, and the peak area data of 14 main common peaks were obtained. The antitussive effect data of 10 batches of Farfarae Flos extract were obtained. Compared with the blank control group, the cough latence in the positive control group and S1, S2, S3, S4, S6, S7, S8, S9, S10 groups was prolonged (all P<0.01), while the cough frequency in 5 minutes in the positive control group and S1, S2, S4, S6, S8, S9, S10 groups was decreased (all P<0.05). The analysis of spectrum-effect relationship revealed that isochlorogenic acid C, isochlorogenic acid A, chlorogenic acid, isochlorogenic acid B, isoquercitrin, and rutin had high contribution to the antitussive effect of Farfarae Flos, and the 6 components were predicted to be the antitussive component group of Farfarae Flos. The verification of bioequivalence showed that there were no statistically significant differences in the antitussive effect between the S9 group and the antitussive component composition group(all P>0.05), which confirmed that isochlorogenic acid C, isochlorogenic acid A, chlorogenic acid, isochlorogenic acid B, isoquercetin, and rutin were the antitussive component group of Farfarae Flos. CONCLUSIONS: The analysis of spectrum-effect relationship combined with the verification of bioequivalence could be used to study the antitussive material basis of Farfarae Flos. The antitussive effect of Farfarae Flos is the result of the joint action of many components.

The phylogeny and divergence time of Ophiocordyceps sinensis and its host insects based on elongation factor 1 alpha.

Ophiocordyceps sinensis Berk. is a fungal parasite that parasitizes the larvae of Hepialidae and is endemic to the Qinghai-Tibet Plateau (QTP). The phylogeny and divergence time of O. sinensis and its host insects were analyzed for 137 individuals from 48 O. sinensis populations based on the elongation factor 1 alpha (EF-1α) gene. Lower nucleotide variation, with only 7 and 16 EF-1α haplotypes, was detected in O. sinensis and its host insects, respectively. The isolated and broad distribution patterns coexisted in both O. sinensis and its host insects on the QTP. The divergence time estimates show that O. sinensis and its host insects originated later than 14.33 million years (Myr) and earlier than 23.60 Myr in the Miocene period, and the major differentiation occurred later than 4 Myr. Their origin and differentiation match well with the second and third uplifts of the QTP, respectively. The host insects from the O. sinensis populations distributed around Qinghai Lake are inferred as an ancient and relict species that has survived various geological events of the QTP. It is suitable to estimate the divergence times of both O. sinensis and its host insects from the same individuals using one gene: EF-1α. Our findings of the origin, phylogeny, and evolution of the endemic species also support the epoch of geological events on the QTP.